The need for transplantation vastly exceeds organ availability, and many of the over 30,000 solid organ transplants (grafts) performed in the United States annually will be lost within 5 years ?primarily as a result of immune-mediated graft rejection. Thus, there is a critical need for improved approaches to reduce graft rejection and to expand the pool of usable organs. A novel strategy for reducing rejection and improving graft quality is to mitigate graft injury occurring prior to transplantation. The majority of transplant organs are from deceased donors, which have inferior outcomes when compared to organs from living donors. This difference is believed to be a result of increased graft injury and immunogenicity caused by inflammation resulting from brain death. The rationale for the proposed research is that identifying the specific cellular and molecular pathways that promote graft rejection in deceased organ donors will lead to the development of novel approaches to improve organ quality prior to transplant. It is now known that mitochondria released into the circulation after brain death are potent stimulators of sterile inflammation and promote graft dysfunction and rejection. The overall objectives of this proposal are to define the specific mitochondria-derived damage associated molecular patterns (mtDAMPs) that cause graft injury, and to develop methods to mitigate mtDAMP-induced inflammation in order to improve graft quality prior to transplantation. The central hypothesis is that the function and survival of transplanted organs can be improved by reducing graft inflammation and injury caused by circulating mitochondria in deceased donors. Guided by strong preliminary data, and using a combination of animal models and human tissues, the hypothesis will be tested through completion of three Specific Aims: 1) Identify the mtDAMPs responsible for increasing organ rejection; 2) Determine the effect of inhibiting mtDAMPs during machine perfusion on graft preservation and post-transplant graft function; and 3) Evaluate the ability of mtDAMP-targeting therapies to reduce human kidney injury during machine perfusion. The results obtained by completing the aims of this proposal will be significant because they will identify specific innate immune pathways responsible for inflammation in deceased organ donors and during ex vivo perfusion. This knowledge will accelerate the development of candidate therapies for abrogating these responses and mitigating graft injury prior to transplant, thus expanding organ utilization and improving organ quality. Treating grafts pre-implantation as a strategy to reduce immune responses following transplant and reduce rates of rejection, or to improve organ quality ex vivo, is innovative, and represents a paradigm shift for strategies aimed at improving transplant outcomes. The knowledge gained though completion of this project will provide a foundation to support subsequent studies, including human clinical trials, with the long-term goal of developing interventions that increase the clinical success of organ transplantation by improving donor organ quality.